1. Field of the Invention
The invention relates to a protein, TSG-6, inducible in connective tissue cells by tumor necrosis factor or interleukin-1, DNA and mRNA encoding the TSG-6 protein, functional derivatives of the protein, antibodies specific to the protein, methods of producing the protein and DNA, and uses of the protein, DNA, mRNA, peptides and antibodies.
2. Description of the Background Art
Tumor necrosis factor (TNF) is a powerful pleiotropic cytokine important in host defenses against tumors and infectious agents. TNF has also been implicated in the pathology of some neoplastic diseases, infections and autoimmune disorders. Most biological actions of TNF can be attributed to the triggering of complex genetic programs in the target cells. Several genes activated by TNF have been identified but many more require characterization.
General Properties of TNF
TNF (also termed TNF-.alpha. and cachectin) is a protein produced by activated monocytes/macrophages which was originally detected in the serum of animals injected sequentially with a bacterial vaccine (bacillus Calmette-Guerin, BCG) and endotoxin (Carswell, E. A. et al., Proc. Natl. Acad. Sci. USA 72:3666 (1975)). TNF is structurally and functionally related to a cytokine produced by activated T lymphocytes which was originally termed lymphotoxin (LT) and is also known as TNF-.beta. (Aggarwal, B. B. et al., J. Biol. Chem. 260:2334 (1985); Williams, T. W. et al., Nature 219:1076 (1968); Ruddle, N. H. et al., J. Exp. Med. 128:1267 (1968); Spies, T. et al., Proc. Natl. Acad. Sci. USA 83:8699 (1986); Gray, P. W. et al., Nature 312:721 (1984); Pennica, D. W. et al., Nature 312:724 (1984)). The genes encoding TNF and LT are linked, and are near the HLA-DR locus on the short arm of human chromosome 6 (Spies, T. et al., supra). TNF and LT bind to common cell surface receptors (Aggarwal, B. B. et al., Nature 318:665 (1985)).
Natural human TNF is a 157 amino acid, non-glycosylated protein with a molecular weight of approximately 17 kDa under denaturing conditions. The mature molecule is derived from a precursor (pre-TNF) which contains 76 additional amino acids at the N-terminus (Pennica, D. W. et al., supra). The expression of the gene encoding TNF is not limited to cells of the monocyte/macrophage family. Several human non-monocytic tumor cell lines were shown to produce TNF (Rubin, B. Y. et al., J. Exp. Med. 164:1350 (1986); Spriggs, D. et al., Proc. Natl. Acad. Sci. USA 84:6563 (1987)). TNF is also produced by CD4.sup.+ and CD8.sup.+ peripheral blood T lymphocytes, and by various cultured T and B cell lines (Cuturi, M. C., et al., J. Exp. Med. 165:1581 (1987); Sung, S.-S. J. et al., J. Exp. Med. 168:1539 (1988)).
Accumulating evidence indicates that TNF is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipase synthesis ("cachectin" activity) (Beutler, B. et al., Nature 316:552 (1985)), activation of polymorphonuclear leukocytes (Klebanoff, S. J. et al., J. Immunol. 136:4220 (1986); Perussia, B., et al., J. Immunol. 138:765 (1987)), inhibition of cell growth or stimulation of cell growth (Vilcek, J. et al., J. Exp. Med. 163:632 (1986); Sugarman, B. J. et al., Science 230:943 (1985); Lachman, L. B. et al., J. Immunol. 138:2913 (1987)), cytotoxic action on certain transformed cell types (Lachman, L. B. et al., supra; Darzynkiewicz, Z. et al., Canc. Res. 44:83 (1984)), antiviral activity (Kohase, M. et al., Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986)), stimulation of bone resorption (Bertolini, D. R. et al., Nature 319:516 (1986); Saklatvala, J., Nature 322:547 (1986)), stimulation of collagenase and prostaglandin E2 production (Dayer, J.-M. et al., J. Exp. Med. 162:2163 (1985)), and other actions. For reviews of TNF, see Beutler, B. et al., Nature 320:584 (1986), Old, L. J., Science 230:630 (1986), and Le, J. et al., Lab. Invest. 56:234 (1987).
TNF also has immunoregulatory actions, including activation of T cells (Yokota, S. et al., J. Immunol. 140:531 (1988)), B cells (Kehrl, J. H. et al., J. Exp. Med. 166:786 (1987)), monocytes (Philip, R. et al., Nature 323:86 (1986)), thymocytes (Ranges, G. E. et al., J. Exp. Med. 167:1472 (1988)), and stimulation of the cell-surface expression of major histocompatibility complex (MHC) class I and class II molecules (Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446 (1986); Pujol-Borrell, R. et al., Nature 326:304 (1987)).
TNF also has various pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J. S. et al., J. Immunol. 136, 1680, 1986)), increased adherence of neutrophils and lymphocytes (Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor (PAF) from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)). Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders (Piguet, P.-F. et al., J. Exp. Med. 166:1280 (1987)), and in cachexia accompanying some malignancies (Oliff, A. et al., Cell 50:555 (1987)). Michie, H. R. et al., Br. J. Surg. 76:670-671 (1989), reviewed evidence that TNF is the principal mediator associated with the pathological changes of severe sepsis.
TNF also has activity associated with growth and differentiation of hemopoietic precursor cells (Murphy, M. et al., J. Exp. Med. 164:263 (1986); Broxmeyer, H. E. et al., J. Immunol. 136:4487 (1986)); some of these actions may be indirect, and are thought to be mediated through the stimulation of production of granulocyte-macrophage colony stimulating factor (GM-CSF) (Munker, R. et al., Nature 323:79 (1986)) and other hemopoietic growth factors (Zucali, J. R. et al., J. Immunol. 140:840 (1988)).
Regulation of Gene Expression by TNF
It is, therefore, apparent that TNF is an extremely "versatile" and clinically significant cytokine. Most of its actions are likely to be mediated by the activation or inactivation of specific genes in the cells upon which it acts. One exception to this mode of action is the rapid cytotoxic effect of TNF on certain target cells; this effect is augmented by inhibitors of RNA or protein synthesis and does not appear to depend on the modulation of gene expression (Matthews, N., Br. J. Cancer 48:405 (1983)). Many specific gene products have been shown to be up-regulated in TNF-treated cells, some of which are discussed below.
Among the first examples of TNF-modulated gene expression was the demonstration that TNF treatment induced an increase in MHC class I mRNA levels and in surface expression of the MHC class I glycoproteins in human vascular endothelial cells (HUVEC) and normal skin fibroblasts (Collins, T. et al., supra). A partial list of other molecules (or genes) induced by TNF appears in Table 1, below. It is interesting to note that TNF is an autoregulatory cytokine, since exogenously added TNF increases TNF synthesis in monocytes and monocytic cell lines (Philip, R. et al., Nature 323:86 (1986); Schmid, J. et al., J. Immunol. 139:250 (1987)).
TABLE 1 GENES AND PROTEINS INDUCED BY TUMOR NECROSIS FACTOR Protein or Gene Ref Cell Type Leukocyte adhesion protein H4/18 HUVEC (1) Platelet-derived growth factor HUVEC and some tumor (2) (PDGF) cell lines IL-6 (IFN-.beta. or BSF-2) Human skin fibroblasts (3) HLA-DR Human tumor cell lines (4) Collagenase Synovial cells and (5) skin fibroblasts 2'-5' oligoadenylate synthetase Tumor cell lines (6) c-myc and c-fos oncogenes Human skin fibroblasts (7) Epidermal growth factor receptor Human skin fibroblasts (8) Tissue factor HUVEC (9) ICAM-1 and ELAM-1 HUVEC (10) Plasminogen activator inhibitors HT1080 cell line (11) 1 and 2 (PAI-1 and PAI-2) Synthesis of 36 kDa and 42 kDa Human skin fibroblasts (12) (=PAI-2) proteins Superoxide Dismutase (MnSOD) gene Human tumor cell lines (13) IL-1.alpha. and IL-1.beta. genes Human skin fibroblasts (14)
REFERENCES: 1. Pober, J. S. et al., J. Immunol. 136, 1680, 1986. 2. Hajjar, K. A. et al., J. Exp. Med. 166, 235, 1987. 3. Kohase, M. et al., Cell 45:659 (19986). 4. Pfizenmaier, K. et al. J. Immunol. 138, 975, 1987. 5. Dayer, J.-M. et al., J. Exp. Med. 162:2163 (1984). 6. Wong, G. H.2. et al., Nature 323:819 (1986). 7. Lin, J.-X. et al., J. Biol. Chem. 262, 11908, 1987. 8. Palombella, V. J. et al., J. Biol. Chem. 262, 1950, 1987. 9. Edgington, T. S. et al., Abs. 2nd. Internat. Conf. TNF, p. 4, 1989. 10. Bevilacqua, M. P. et al., Proc. Natl. Acad. Sci. USA 84, 9238, 1987. 11. Medcalfe, R. L. et al., J. Exp. Med. 168, 751, 1988. 12. Kirstein, M. et al., J. Biol. Chem. 261, 9565, 1986. 13. Wong, G. H. et al., Science 242, 941, 1988. 14. Le, J. et al. Lab. Invest. 56:234 (1987).
The inhibitory actions of TNF on gene expression are less well-characterized. TNF was shown to inhibit c-myc expression in cells whose growth it inhibited (Kronke, M. et al., Proc. Natl. Acad. Sci. USA 84:469 (1987)). Collagen synthesis was inhibited in human fibroblasts (Solis-Herruzo et al., J. Biol. Chem. 263:5841 (1988)), and thrombomodulin in HUVEC (Conway, E. M. et al., Molec. Cell. Biol. 8:5588 (1988)). All these inhibitory actions were expressed at the level of transcription, but the precise mechanisms are still unclear.
The mechanisms of signal transduction and gene activation by TNF are the subject of great interest. In many cell types, TNF activates a phospholipase (most likely PLA2), resulting in the liberation of arachidonic acid from cellular pools (Suffys, P. et al., Biochem. Biophys. Res. Comm. 149:735 (1987)) and increased eicosanoid synthesis (Dayer, J.-M. et al., supra). In human fibroblasts, TNF stimulated GTPase activity (Imamura, K. et al. J. Biol. Chem. 263:10247 (1989)), raised cAMP levels, enhanced cAMP-dependent protein kinase activity, and activated protein kinase C (PKC) (Zhang, Y. et al., Proc. Natl. Acad. Sci. USA 85:6802 (1988); Brenner, D. A. et al., Nature 337:661 (1989)). TNF can also activate the transcription factor NF-kB, which appears to be the mechanism by which TNF induces the IL-2 receptor .alpha. chain (Lowenthal, J. W. et al., Proc. Natl. Acad. Sci. USA 86:2231 (1989)) or cause activation of latent human immunodeficiency virus, HIV-1 (Griffin, G. E. et al., Nature 339:70 (1989)).
Interactions of TNF with other Cytokines
When the individual actions of TNF-.alpha., TNF-.beta., IL-1.alpha., IL-1.beta., IFN-.alpha., IFN-.beta. or IFN-.tau. are compared in various experimental systems, a great deal of apparent redundancy and ambiguity is noted. First, structurally related cytokines which utilize the same receptor (e.g., TNF-.alpha. and TNF-.beta.; IL-1.alpha. and IL-1.beta.; IFN-.alpha. and IFN-.beta.) act similarly. More surprisingly, structurally unrelated cytokines which bind to different receptors also have similar physiological effects. For example, IL-1 and TNF have similar gene activating activities, and result in similar biological effects (Le, J. et al. Lab. Invest. 56:234 (1987)). IFNs and TNF also share biological activities (Kohase, M. et al., Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986); Williamson, B. D. et al., Proc. Natl. Acad. Sci. USA 80:5397 (1983); Stone-Wolff, D. S. et al., J. Exp. Med. 159:828 (1984)). For example, IFNs and TNF activate some of the same genes, including MHC class I and class II genes, 2'-5' oligo-adenylate synthetase, IL-6, the transcription factor IRF-1, and the TNF gene itself (Vilcek, J., Handbook of Experimental Pharmacology, Vol. 95/II, p. 3, Springer-Verlag, Berlin (1990)).
Under natural conditions, cells are rarely, if ever, exposed to a single cytokine. Rather, cytokine action in vivo is "contextual," as has been postulated for growth factors (Sporn, M. B. et al., Nature 332:217 (1988)). The biological effects produced by cytokines under natural conditions must therefore represent the sum of the synergistic and antagonistic interactions of all cytokines present simultaneously in a given microenvironment. In addition, cytokines appear to be arranged in "networks" and "cascades", such that the synthesis of one cytokine can be positively or negatively regulated by another. For these reasons, it is important to understand the molecular mechanisms of action of cytokines acting individually as well as in combination.
In contrast to the above, there are cases in which the actions of TNF and IFNs are antagonistic rather than similar or synergistic. For example, TNF is mitogenic for human diploid fibroblasts, whereas IFNs inhibit growth of these cells (Vilcek, J. et al., J. Exp. Med. 163:632 (1986)). The cellular response to a combination of TNF and an IFN can differ from the response to either one alone, both qualitatively and quantitatively (Leeuwenberg, J. F. M. et al., J. Exp. Med. 166:1180 (1987); Reis, L. F. L. et al., J. Biol. Chem. 264:16351 (1989); Feinman, R. et al., J. Immunol. 136:2441 (1986); Trinchieri, G. et al., Abstr. 2nd Int'l Conf. TNF, p. 7 (1989)). To make matters even more complicated, in some cells TNF can induce IFN-.beta. synthesis (Reis et al., supra); the activation of some genes (e.g., HLA class I) by TNF requires the presence of IFN-.beta. (Leeuwenberg et al., supra). Since IFNs and TNF-.alpha. and TNF-.beta. are often produced in the same microenvironment in response to a similar set of stimuli (Murphy, M. et al., supra; Stone-Wolff et al., supra; Billiau, A., Immunol. Today 9:37 (1988)), it is clear that the interactions of TNF and IFNs are highly relevant to the outcome in vivo under either "normal" or pathophysiological conditions.
The association of cytokines, in particular TNF, with cancer and infectious diseases takes many forms often related to the host's catabolic state. One of the major and most characteristic problems seen in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results is known as "cachexia" (see, for review, Kern, K. A. et al. (J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth. The fundamental physiological derangement may be related to a decline in food intake relative to energy expenditure. The causes for this commonly observed and often life-limiting disturbance remain to be determined, even though many contributing factors have been identified (Braunwald, E. et al. (Eds.), Harrison's PRINCIPLES OF INTERNAL MEDICINE, 11th Ed., McGraw-Hill Book Co., New York, 1987, Chap. 78, pp. 421-431). The cachectic state is associated with significant morbidity and is responsible for the majority of cancer mortality. A number of studies have suggested that TNF is an important mediator of the cachexia in cancer, infectious disease, and in other catabolic states.
It has been known for some time that in bacterial infection, sepsis and critical illness, bacterial lipopolysaccharides (LPS), or endotoxins, are responsible for many of the pathophysiological manifestations, including fever, malaise, anorexia, and cachexia. More recently, it was observed that TNF can mimic many endotoxin effects, leading to the suggestion that TNF, and related cytokines derived from cells of the macrophage/monocyte family, in particular, IL-1, are central mediators responsible for the clinical manifestations of the illness. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kornbluth, S. K. et al., J. Immunol. 137:2585-2591 (1986)) and other cytokines including IL-1 (Dinarello, C. A., Rev. Infec. Dis. 6:51-94 (1984)), interleukin-6 (IL6), and colony stimulating factor (CSF) (Apte, R. N. et al. J. Cell. Physiol. 89:313 (1976)). Some of these cytokines further stimulate T lymphocytes to produce additional cytokines, for example, interleukin-2 (IL-2) (Robb, R. J., Immunol. Today 5:203-209 (1984)).
The monocyte-derived cytokines are thought to be important mediators of the metabolic and neurohormonal responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med. 318:1481-1486 (1988)), and in cancer and other catabolic states (Norton, J. A. et al., Nutrition 5:131-135 (1989)). Interestingly, some changes induced by low-dose TNF closely resemble changes provoked by high dose IL2 (Remick, D. G. et al., Lab. Invest. 56:583-590 (1987)).
Endotoxin administration to human volunteers produced acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 123:162-170 (1988)). Treatment of cancer patients (having normal kidney and liver function) with escalating doses of TNF (4-636 .mu.g/m.sup.2 /24 hr) indicated that doses greater than 545 .mu.g/m.sup.2 /24 hr caused alterations similar to those induced by injection of endotoxin (4 ng/kg) into healthy humans (Michie, H. R. et al., Surgery 104:280-286 (1988)), leading the authors to conclude that TNF is the principal host mediator of septic and endotoxemic responses. More recently, it was shown that five days of chronic intravenous TNF infusion into humans or rats was associated with anorexia, fluid retention, acute phase responses, and negative nitrogen balance (i.e., classic catabolic effects), leading to the conclusion that TNF may be responsible for many of the changes noted during critical illness (Michie, H. R. et al., Ann. Surg. 209:19-24 (1989)). Administration of rTNF to cancer patients also led to a rise in C-reactive protein (CRP) and a fall in serum zinc, a large increase in forearm efflux of total amino acids, and amino acid uptake by other tissues (Warren, R. S. et al., Arch. Surg. 122:1396-1400 (1987)), considered further evidence for a role of TNF in cancer cachexia.
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